Light Microscopy

Question 1

What size can the eye see down to

Answer 1

0.2mm

Question 2

Light microscopy can see down to

Answer 2

0.2um

Question 3

Electron microscope can see down to

Answer 3

0.2nm

Question 4

Magnification

Answer 4

Ratio of size of image to object

Total = power of objective lens x power of eyepiece

Question 5

Resolution

Answer 5

Clarity of image, illumination and quality of optics

Question 6

Resolution (D) =

Answer 6

(0.61 x wavelength)/NA
NA = N x sina
NA is numerical aperture and labelled on the objective lens
a is angular apeture and N is the refractive index of the medium between the specimen and objective lens
Smaller D = better resolution

Question 7

Contrast

Answer 7

contrast between lightest and darkest area of sample

Question 8

Numerical aperture

Answer 8

size of cone of light coming from each point of the specimen

Question 9

Hooke

Answer 9

First named a cell

Simple microscope with oil lamp as a light source

Question 10

Leeuwenhoek

Answer 10

improved production of lenses and at over 200 x magnification observed cell types

Question 11

Zeiss

Answer 11

Made lenses allowing resolution at theoretical limits

Question 12

Components of light microscopy

Answer 12

Objective lens,( collects cone of light rays to make an image), condenser lens (focuses cone of light rays on specimen), eyepiece and light source

Question 13

Brightfield light microscopy

Answer 13

Poor images of living cells compared to phase contrast and DIC

Question 14

Phase contrast

Answer 14

High contrast images
density of specimen determines diffraction of light
Diffracted and undiffracted rays give rise to changes in brightness- contrast
Degree of darkness/brightness depends on refractive index of a region
Refracted and unrefracted light recombine at image plane to form the image

Question 15

DIC

Answer 15

use polarised light - polariser
Prism splits source of light into 2 parts, 2nd prism recombines
If one of beams is diffracted by the specimen, when they are recombined they will interfere and generate contrast ranging from black to white
If beams not diffracted they don’t interfere when recombined and produce pale grey
Contrast generated by difference in refractive index of specimen and surrounding medium

Question 16

Tissue sectioning

Answer 16

cut into thin slices

Question 17

Detection of cellular components/ viability with bright field using:

Answer 17

dyes
chromogenic (production of colour or pigments) enzyme substrate
Dyes

Question 18

Hysto or cyto chemical dyes

Answer 18

Give rise to rust colour

Interact with phenolic compounds in cells and lignin in CWs

Question 19

Which types of microscopy can be used in time lapse microscopy

Answer 19

Phase contrast and DIC - produces movie through multiple images

Question 20

Fluoresence

Answer 20

Absorbing light of a particular wavelength then emitting light of a different colour and wavelength

Question 21

fluoresence vs bright field

Answer 21

Main difference is use of selective filters and directing excitation light through objective lens, into the sample and observing emitted fluorescent light passing back through objective lens from sample.
Excitation light reflected onto dichroic mirror

Question 22

Applications of fluorescence microscopy

Answer 22

visualising organelles
Different dyes for relative localisation of molecules
Autofluorescence can reveal certain cell components
Immunofluorescence microscopy used to detect specific proteins with an antibody to which a fluorescent dye has been covalently attached

Question 23

Confocal microscopy

Answer 23

limitation of fluorescence: fluorescent light come not only from plane of focus but also molecules above and below- see blurred image due to fluorescent images from many depths
Better resolution because only plane in focus is visible
More advanced, images in multiple planes
Image live specimens, take images at multiple intervals

Question 24

Uses of confocal

Answer 24

used in co-localization, intracellular, thick specimens

Question 25

Co-localization

Answer 25

observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different “targets” are located in the same area of the cell or very near to one another.